Human recombinant hcd40l

Primary human B cells were cultured in IMDM medium (Life Technologies, Carlsbad, CA, USA) containing 10% heat-inactivated foetal calf serum, 2mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid buffer (HEPES), 1 mM sodium pyruvate and 50 μM 2-mercaptoethanol (all from Invitrogen, Carlsbad, CA, USA). Human IL2 (20 ng/ml; R&D Systems, Minneapolis, MN, USA) and human IL4 (20 ng/ml; R&D) were immediately added as supplements prior to initiating the experiments. Where indicated, human recombinant hCD40L (250 ng/ml; R&D Systems) and 25 μM CpG-ODN 2006 (InvivoGen, San Diego, CA, USA) were used. The cells were cultured at 1 × 10⁶ cells/ml in either 24-well (1 ml) or 48-well culture plates (0.5 ml).

TMC were cultured in IMDM medium (Life Technologies, CA, USA) containing 10% heat-inactivated fetal calf serum, 2mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES), 1 mM sodium pyruvate and 50 µM 2-mercaptoethanol (all from Invitrogen, CA, USA). Human IL2 (20 ng/ml; R&D Systems, MN, USA) and human IL4 (20 ng/ml; R&D Systems, MN, USA) were added immediately before experiments also as supplements. When indicated, human recombinant hCD40L (250 ng/ml; R&D Systems, MN, USA) and 25 µM CpG-ODN 2006 (InvivoGen, CA, USA) were used. Cells were cultured at 1x10⁶ cells/ml either in 24-well culture plates (1ml) or 48-well culture plates (0.5ml).